Specificity of ethephon as a butyrylcholinesterase inhibitor and phosphorylating agent.
Specificity of ethephon as a butyrylcholinesterase inhibitor and phosphorylating agent.
复制标题
乙烯利作为丁酰胆碱酯酶抑制剂和磷酸化剂的特异性。
DOI：
10.1021/tx020042w
复制
发表时间：
2002
影响因子：
4.1
通讯作者：
Casida,JohnE
中科院分区：
文献类型：
--
作者：
Haux,JEric;Lockridge,Oksana;Casida,JohnE
植物生长调节剂（2-氯乙基）膦酸（乙烯利）可抑制丁酰胆碱酯酶（BChE），25年前在大鼠和小鼠的体外和体内试验中观察到了这一点，最近在对人类受试者进行的低剂量亚慢性研究中也观察到了这一点。推测其作用机制是乙烯利二价阴离子对BChE活性位点S198的磷酸化。本研究测试这一假设直接使用[33 P]乙烯利和重组BChE（rBChE）与单个氨基酸取代，并进一步评估，如果BChE是最敏感的酯酶靶在体外和小鼠体内。[33 P]乙烯利标记纯化的rBChE，但不标记无酶活性的二乙基磷酰-rBChE（通过与毒死蜱氧磷预孵育在S198处衍生化）或其他几种酯酶和蛋白质。氨基酸取代大大降低了rBChE对乙烯利的敏感性，是氧阴离子孔中的G117 H和G117 K（可能干扰甘氨酸-N-H和乙烯利二价阴离子之间的氢键）和A328 F，A328 W和A328 Y（可能阻碍进入活性位点峡谷）。其他不影响灵敏度的取代是D 70 N、D 70 K、D 70 G和E197 Q，它们不直接参与催化三联体。pH值和缓冲液组成对抑制的影响支持了乙烯利二价阴离子是实际磷酸化剂而不被二价阳离子活化的假设。人血浆BChE在体外和小鼠血浆BChE在体外和体内是更敏感的乙烯利比任何其他酯酶检测丁酰硫代胆碱或1-乙酸萘酯水解在native-PAGE。在体外和体内，观察到的所有小鼠肝酯酶对乙烯利的敏感性均低于血浆BChE。其他十几种酯酶对乙烯利的敏感性比BChE低10 - 100倍。因此，BChE抑制仍然是乙烯利暴露最敏感的标志。
Butyrylcholinesterase (BChE) is inhibited by the plant growth regulator (2-chloroethyl)phosphonic acid (ethephon) as observed 25 years ago both in vitro and in vivo in rats and mice and more recently in subchronic studies at low doses with human subjects. The proposed mechanism is phosphorylation of the BChE active site at S198 by ethephon dianion. The present study tests this hypothesis directly using [33P]ethephon and recombinant BChE (rBChE) with single amino acid substitutions and further evaluates if BChE is the most sensitive esterase target in vitro and with mice in vivo. [33P]Ethephon labels purified rBChE but not enzymatically inactive diethylphosphoryl-rBChE (derivatized at S198 by preincubation with chlorpyrifos oxon) or several other esterases and proteins. Amino acid substitutions that greatly reduce rBChE sensitivity to ethephon are G117H and G117K in the oxyanion hole (which may interfere with hydrogen bonding between glycine-N−H and ethephon dianion) and A328F, A328W, and A328Y (perhaps by impeding access to the active site gorge). Other substitutions that do not affect sensitivity are D70N, D70K, D70G, and E197Q which are not directly involved in the catalytic triad. The effect of pH and buffer composition on inhibition supports the hypothesis that ethephon dianion is the actual phosphorylating agent without activation by divalent cations. Human plasma BChE in vitro and mouse plasma BChE in vitro and in vivo are more sensitive to ethephon than any other esterases detected by butyrylthiocholine or 1-naphthyl acetate hydrolysis in native−PAGE. All mouse liver esterases observed are less sensitive than plasma BChE to ethephon in vitro and in vivo. More than a dozen other esterases examined are 10−100-fold less sensitive than BChE to ethephon. Thus, BChE inhibition continues to be the most sensitive marker of ethephon exposure.